1. Field of the Invention
This invention relates to the quantitative determination of substances in or characteristics of liquid media, including body fluids such as serum, based on specific binding assay techniques. In particular, the invention is directed to the detection of antigens or haptens based on immunoassay techniques involving the use of labeled reagents, such as radiolabeled reagents. The present invention provides an improved method of performing the separation of bound- and free-label inherent in heterogeneous specific binding assays.
2. Description of the Prior Art
A living system is able to detect, recognize and respond to the presence of foreign material (antigen) such as protein, virus, bacteria, and so forth, within that system. This response takes, inter alia, the form of producing an antibody specific for the particular antigen. There then occurs a specific reaction between the antibody and the antigen to form a complex. An antibody once produced is also capable of binding a hapten, i.e., a relatively small and simple compound which may be the determinant group of a given antigen, which hapten is capable of binding with the specific antibody but incapable itself of giving rise to the production of an antibody, unless it is bound to an antigenic carrier.
The binding interaction between an antigen or a hapten and its antibody is specific and sensitive. Other types of materials that participate in similar specific and sensitive binding interactions are enzymes and their substrates; materials such as hormones, vitamins, metabolites and pharmacological agents, and their receptors and binding substances; and other substances known in the science. These specific and sensitive binding reactions have given rise to a rapidly emerging analytical technique known as the specific binding assay technique. In one such type of assay method, the substance, or group of substances, to be determined (herein referred to as "ligand") in a liquid sample is placed in competition with a labeled form of the ligand or of a binding analog thereof for binding to a binding reagent. Where a radioactive label is used and the binding reagent is an antibody, the method is known as a radioimmunoassay method. Recently, several alternative labeling materials have been reported for replacement of radioisotopes, including enzymes, coenzymes, enzyme substrates, enzyme modulators such as inhibitors and allosteric effectors, fluorescent molecules, luminescent molecules, and others. For illustrative purposes, the discussion which follows describes one particular type of specific binding assay technique, a competitive binding radioimmunoassay technique.
This system consists of antigen or hapten labeled with a radioactive marker, unlabeled native antigen (in the test sample) and specific antibody whereby there is competition between the unlabeled antigen and the labeled antigen for binding to a limited amount of antibody. Hence, the greater the concentration of unlabeled antigen from the test sample in the system, the less the labeled antigen will be bound by the antibody. This may be diagrammatically represented as follows: ##STR1##
If the concentration of labeled antigen and antibody is fixed and the only variable is the level of unlabeled antigen, it becomes possible to establish an assay system for measuring the unknown level of unlabeled antigen by physically separating the antigen-antibody complex from the remaining free antigen (both labeled and unlabeled). The radioactivity of the unknowns is compared with a standard curve plotting of the values given by a range of known amounts of the antigen treated in the same manner.
The need for a simple and reliable method for accomplishing the essential separation of the bound- and free-species of the labeled component in heterogeneous specific binding assays has been felt for many years, ever since the analytical value of such assays was fully realized. Numerous separation techniques have evolved including chromatoelectrophoresis, a cumbersome technique; ascending paper-wick chromatography [Scand. J. Clin. Lab. Invest. 20:297(1967)], a limited and commercially unattractive technique; precipitation of the antigen-antibody complex, a technique requiring the use of a centrifuge and which in some cases yields variable results due to the lack of selectivity in the precipitating agents available; the double-antibody technique, requiring an additional antibody reagent and additional incubation time; and the use of solid phase binders, such as solid phase antibodies and various adsorbents, all of which have left room for improvement.
Recently, there has been developed a novel separation technique referred to as the ascending column chromatography technique which is described in U.S. Pat. No. 4,205,058 and assigned to the instant assignee. In accordance with this new method, the bound-species and free-species of the labeled component are separated by contacting at least a portion of the binding reaction mixture, a predetermined time after its formation, with a column comprising an adsorbent material which is both selective for binding one of said bound- and free-species and capillarily absorbent relative to said reaction mixture whereby said reaction mixture is drawn into the column by capillary action and said bound- and free-species are separated along the column. This technique provided for the first time a practical, fast and reliable method of separation which is effected passively, without any significant care required of the technician performing the assay. After incubation of the binding reaction mixture, the technician simply places an adsorbent column in contact with the mixture and waits until the mixture has been drawn into the column, at which time separation is inherently accomplished by the selective binding capacity of the adsorbent. The adsorbed species is held at the beginning portion of the column, while the nonadsorbed species is transported by capillary migration of the liquid carrier toward the other end of the column. The label, usually a radioactive material, can then be measured in one or the other of the separated species by measurement at the beginning portion or terminal portion of the column.
While in principle the ascending column chromatography technique is applicable to the determination of any ligand in, or any ligand binding capacity of, a liquid medium, certain unforeseen difficulties have been encountered in some applications of the technique. For example, since it is most desirable to achieve as great and sharp a spatial separation of the bound- and free-species as possible along the column, it is generally desirable to select an adsorbent whose affinity for binding one of such species (in practice, usually the free-species) is quite high. However, it has been unexpectedly found that sometimes this affinity can be so high that it creates a competition between the ligand binding agents present in the reaction mixture and the adsorbent for binding of the ligand. As a result, the complexes of labeled and unlabeled ligand or binding analog bound to binding agent which had formed during the binding reaction are dissociated and the released labeled material, converted into a free-species of the labeled component, becomes adsorbed to the column material. As this process occurs during the capillary migration of the reaction mixture along the column, there results a "trailing" of labeled component along the column, thereby reducing the sensitivity and reproducibility of the assay. Such trailing effect is particularly troublesome where radiolabels are used and no radiopaque shield is used in measuring the label in one of the separated species as described in the aforementioned U.S. patent application. Other difficulties encountered in certain applications of the previously described ascending column chromatography technique to which the present invention is addressed are discussed in the disclosure to follow.
It is, therefore, a principal object of the present invention to provide an improved specific binding assay of the above-described "ascending column chromatography" type, wherein the above described undesirable phenomenon of "trailing" of labeled material along the column is eliminated so as to afford a sharper and better separation between the bound- and free-species. It is a further object of the invention to provide a specific binding assay method of the above-mentioned type wherein, for a given reaction system involving a particular ligand and its binding partner, the selectively adsorbent material may be chosen from a broader range of materials, including those which hitherto have been excluded from use in the known ascending column chromatography technique because of excessive affinity for either the ligand or its binding partner. Yet another object of the invention is to provide an ascending column chromatography specific binding assay which permits the use of considerably smaller amounts of the selectively adsorbent material, the cost of which is, in some cases, an important factor in determining the practical and commerical application of the method.